CN103374606B - CHEK1 (checkpoint kinase 1) gene mutation detection specific primers and liquid chip - Google Patents
CHEK1 (checkpoint kinase 1) gene mutation detection specific primers and liquid chip Download PDFInfo
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Abstract
The invention discloses a CHEK1 (checkpoint kinase 1) gene mutation detection liquid chip and specific primers. The liquid chip mainly comprises each ASPE (allele specific primer extension) primer formed by tag sequences at 5' terminal and specific primer sequences aiming at target gene mutation sites at 3' terminal, microspheres coated by anti-tag sequences as well as amplification primers, wherein the specific primer sequences are SEQ ID NO.9 and SEQ ID NO.10 aiming at C163T sites, SEQ ID NO.11 and SEQ ID NO.12 aiming at C98A sites, SEQ ID NO.13 and SEQ ID NO.14 aiming at G106A sites and/or SEQ ID NO.15 and SEQ ID NO.16 aiming at G106A sites. The liquid chip has the advantages that the coincidence rate of the detection results of the detection liquid chip provided by the invention and a sequencing method is as high as 100%; and parallel detection of wild types and mutant types of a plurality of mutation sites is achieved.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a CHEK1 gene mutation detection specific primer and a liquid phase chip.
Background
Checkpoint kinase 1(Checkpoint kinase 1, CHEK1) is a serine/threonine protein kinase playing a key role in mediating the cellular response of DNA damage, and a gene CHEK1 encoding the protein is located on chromosome 11q24-q24, and the gene CHEK1 is a cell cycle regulatory Checkpoint gene related to DNA damage repair, mainly a DNA damage Checkpoint at the S phase of cells, namely CHEK1 plays a Checkpoint role at the S phase. CHEK1 is a tumor suppressor gene that protects the body's genome from mutations, protects the body from cancer, or otherwise protects the body from cancer, but it also promotes cancer cell growth while protecting the body from cancer. That is, the CHEK1 gene can restrict spontaneous mutation of other genes in cells to prevent cancer, but when the generated cancer shows enlarged damage to the body's own DNA, the CHEK1 gene can indirectly help tumor cells by reducing damage of genome, and the CHEK1 can prevent damage of replication stress (replication stress), but when the damage occurs in the genetic material during cell division, some tumor cells can indeed cause continuous damage in genome to result in high rate of damage. At present, research shows that the CHEK1 gene mutation is related to the occurrence and development of lung cancer and pancreatic cancer, and the practical application guided by the detection of the mutation condition becomes a research hotspot in the field.
At present, the CHEK1 gene mutation detection method mainly comprises the following steps: PCR-RFLP, direct sequencing and fluorescent quantitative PCR technology, wherein the PCR-RFLP method is based on the change of restriction enzyme recognition sites caused by gene mutation, such as site loss or new site generation, a specific fragment is amplified through PCR, the amplified product is cut by restriction enzyme, and the size of the fragment is observed through electrophoresis. Other detection technologies based on PCR, such as direct sequencing and fluorescent quantitative PCR, have the disadvantages of low sensitivity, easy sample contamination and high false positive rate. Thirdly, the methods have the limitation of detection flux, only one mutation type can be detected at a time, and the requirements of practical application cannot be met.
The CHEK1 gene mutation sites detected by the target of the invention are shown in the table:
| serial number | Content of CHEK1 gene site mutation | Shorthand writing |
| 1 | Nucleotide 163 of SEQ ID NO.39, the C → T mutation | C163T |
| 2 | Nucleotide 98 of SEQ ID NO.40, the C → A mutation | C98A |
| 3 | Nucleotide 106 of SEQ ID NO.41, the G → A mutation | G106A |
| 4 | Nucleotide 159 of SEQ ID NO.41, the A → G mutation occurred | A159G |
SEQ ID NO.39 C163T
GACAGGGTCTTACCATGTTGGCCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCGCCCACCCCAG
CCTCCCAAAAAGCTGGGATTACAGGTGTGAGCCACAGCCTATATTTAAAAAATATACTAATGATAGCT
TTTACTCAAATATTTAAATTCTTCTTAGGAAAAGGTATAATGAAAATCAATGAAACACACCTGATTC
ATACAACTTTTCTTCCATTGATAGCCCAACTTCTCACAAGTCTCTTTCAGGCATTGATAAGATTTGTC
TGCATCCAATTTGGTAAAGAATCGTGTCATTCTTTTGACCCACCGCTGCCAGGGGTTCTACATGT.
SEQ ID NO.40 C98A
CATCCGGTGTGAGGCTCGACTGGGCGGTGCTCGAGCGCTGCTGGGGGAAGCCCAGGCCTCCAAGCCTC
AGGGGCGCGCGACCGACCGCGCTGCGCTTCAGGCGGGGCTGCGGGCTTCGCTCCTGGCCGCTCTGGG
CCTGAAAGGCTGCACCGCCACCTGCGTGAGGCCACCGGACAGGACTGTGAGGATCACAGTCGGTGAAG
CAGAGTGCTTTGTAAACCTCAGAGTGCGGTACTCGTGGTGCTGG。
SEQ ID NO.41 G106A、A159G
GCCGAGAAACCAGCAGCAGCTGCTGAGAGCCGACTGTGAAGAAATGGGAGGAACTCCGTGTTGGGAGC
GAGAGGCTTCGAAAGAACTGGCCACGCAGTCAAGTGTTGTGGCGGGGCAAGTTTCCCGGAGAAAGCG
AGCAGTTTATGTTGGAGCTTGAGTCAGGATAACCAGTTTTGCCTTTTTTCTGTGAACCAGCTCCAAA
ACAATGCTTTACTTCAGCCCGGTCTTTTTGCAGGAAGACCATCTGCTTAGCTTAGTTCTACAAACTTT
TTCATTTTTAATGTGCAAAGAACAGGCGGGGAGAGCCAAAATAAATCTTACAACGCAAATTTAAAAGC
TGCGTACGTCATTTAAGTGGA。
Disclosure of Invention
One of the purposes of the invention is to provide a liquid phase chip for detecting CHEK1 gene mutation, which can be used for detecting wild type and mutant type of four common genotypes of C163T, C98A, G106A and A159G of CHEK1 gene individually or in parallel.
The technical scheme for realizing the purpose is as follows:
a liquid phase chip for detecting CHEK1 gene mutation comprises:
(A) wild-type and mutant ASPE primer pairs designed for different mutation sites of the CHEK1 gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: SEQ ID NO.9 and SEQ ID NO.10 for the C163T site, SEQ ID NO.11 and SEQ ID NO.12 for the C98A site, SEQ ID NO.13 and SEQ ID NO.14 for the G106A site, and/or SEQ ID NO.15 and SEQ ID NO.16 for the A159G site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 8;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) primers for amplifying a target sequence to be detected having a corresponding mutation site. In some of these embodiments, the amplification primers are: SEQ ID NO.25 and SEQ ID NO.26 for the C163T site, SEQ ID NO.27 and SEQ ID NO.28 for the C98A site, and/or SEQ ID NO.29 and SEQ ID NO.30 for the G106A site, A159G site.
In some of these embodiments, the ASPE primers are: the sequence consisting of SEQ ID NO.1 and SEQ ID NO.9 and the sequence consisting of SEQ ID NO.2 and SEQ ID NO.10 for the C163T site, the sequence consisting of SEQ ID NO.3 and SEQ ID NO.11 and the sequence consisting of SEQ ID NO.4 and SEQ ID NO.12 for the C98A site, the sequence consisting of SEQ ID NO.5 and SEQ ID NO.13 and the sequence consisting of SEQ ID NO.6 and SEQ ID NO.14 for the G106A site, and/or the sequence consisting of SEQ ID NO.7 and SEQ ID NO.15 and the sequence consisting of SEQ ID NO.8 and SEQ ID NO.16 for the A159G site.
Another objective of the invention is to provide specific primers for CHEK1 gene mutation detection.
The technical scheme for realizing the purpose is as follows:
specific primers for CHEK1 gene mutation detection, wherein the specific primer sequences are as follows: SEQ ID NO.9 and SEQ ID NO.10 for the C163T site, SEQ ID NO.11 and SEQ ID NO.12 for the C98A site, SEQ ID NO.13 and SEQ ID NO.14 for the G106A site, and/or SEQ ID NO.15 and SEQ ID NO.16 for the A159G site.
The main advantages of the invention are:
1. the coincidence rate of the detection result of the liquid phase chip for detecting the gene mutation of the CHEK1 provided by the invention and a sequencing method is up to 100%, and the required time is far lower than that of a common sequencing technology, thereby particularly meeting the requirement of practical application. The prepared liquid phase chip for detecting the mutation of the CHEK1 gene has very good signal-to-noise ratio, no cross reaction exists between the designed probe and the anti-tag sequence basically, the tag sequence, the anti-tag sequence are selected, and the tag sequence is combined with a specific ASPE primer, so that the cross reaction can be avoided, and the parallel detection of a plurality of mutation sites can be realized.
2. The ASPE primer specific primer designed by the invention can sensitively and specifically identify the mutation site of target detection and accurately distinguish genotypes of various types; in the same reaction system, cross reaction basically does not exist among different specific primers and between the specific primers and a PCR amplification product for non-target detection, the detection specificity is good, and the cross reaction rate is lower than 3%; besides the mutation condition of a single site, the mutation condition of a plurality of mutation sites can be simultaneously detected in parallel, and the detection effects are consistent.
3. The invention not only overcomes the defects of low sensitivity and poor repeatability of detection results of the traditional solid phase chip, but also improves the existing liquid phase chip technology, so that the prepared microspheres can be suitable for different detection items and have strong expansibility. The detected fluorescence signal value is greatly improved, so that the detection sensitivity is further improved, the signal-to-noise ratio is enhanced, and the detection result is more accurate and reliable.
4. In addition, the detection method of the invention has simple steps, and the 4 mutation sites can be detected by one-step PCR to complete the amplification of 3 target sequences containing the mutation sites, thereby avoiding a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like, greatly improving the detection accuracy and embodying the accurate and simultaneously qualitative and quantitative analysis characteristics.
Detailed Description
Example 1
The liquid phase chip for detecting the gene mutation of the CHEK1 mainly comprises the following components:
first, ASPE primer
Specific primer sequences are designed aiming at wild types and mutant types of four common genotypes of the CHEK1 gene, namely C163T, C98A, G106A and A159G respectively. The ASPE primer consists of a tag sequence and a specific primer sequence. The ASPE primer sequences are shown in the following table:
TABLE 1 ASPE primer sequences (tag sequence + specific primer sequence) of CHEK1 Gene
Each ASPE primer comprises two parts, wherein the 5 'end is a specific tag sequence aiming at an anti-tag sequence on a corresponding microsphere, and the 3' end is a mutant type or wild type specific primer segment (as shown in the table 1). All ASPE primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Two, anti-tag sequence coated microsphere
According to the designed ASPE specific primer fragment, tag sequences are selected, secondary structures possibly formed among anti-tag sequences of the microspheres and between tag and the ASPE specific primer fragment are reduced to the maximum extent, and the number of 8 selected microspheres and the corresponding anti-tag sequences on the microspheres are shown in Table 2:
TABLE 2 numbering of microspheres and corresponding anti-tag sequences on microspheres
The 8 selected microspheres were purchased from Luminex, USA, and the anti-tag sequence was coated on the microspheres. 5-10T spacer arm sequences are connected between the anti-tag sequences and the microspheres, namely a 5-10T spacer arm sequence is added in front of each anti-tag sequence, and the anti-tag sequences are synthesized by Shanghai Bioengineering technology service GmbH. The synthetic anti-tag sequence was treated with sterile ddH2O is prepared into a 100nmol/ml stock solution. The spacer arm is used to separate the anti-tag from the surface of the microsphere or to separate the anti-tag from the surface of the microspheretag is a sequence placed in a hydrophilic environment. By arranging a spacer arm sequence with proper length between the anti-tag sequence and the microsphere, the steric hindrance can be reduced, and the efficiency of hybridization reaction and the specificity of the hybridization reaction are improved. Common spacer sequences include poly-dT, i.e., poly (dT), oligo-tetrapolyethylene glycol, and (CH2) n spacers (n.gtoreq.3), such as (CH2)12, (CH2)18, and the like. In addition, if a poly (dA) interference is present, poly (TTG) may also be used as a spacer. The spacer arm of the invention is preferably 5-10T, and the process of coating the microspheres is as follows: respectively taking 5 × 106Each of the above numbered carboxylated microspheres (from Luminex) was suspended in 50ul of 0.1mol/L MES solution (pH4.5) and 10ul of synthetic anti-tag molecule (100nmol/ml) was added. 10ng/ml of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) working solution (available from Pierce Chemical Co.) 2.5ul of EDC working solution was added to the microsphere suspension, incubated at constant temperature for 30 minutes, 2.5ul of EDC working solution was added, incubated at constant temperature for 30 minutes, washed once with 0.02% Tween-20 and then 0.1% SDS after the completion of the reaction, and the washed microspheres coated with the anti-tag sequence were resuspended in 100ul of Tris-EDTA solution [10mmol/L Tris (pH8.0) ]]In 1mmol/L EDTA, storing at 2-8 deg.c in dark.
Thirdly, amplifying the primer of the target sequence containing the mutation site
Aiming at four common genotypes of the CHEK1 gene, namely C163T, C98A, G106A and A159G, an amplification primer pair (see Table 3) is designed, wherein G106A and A159G are positioned in the same amplification product, and three target sequences containing mutation sites are respectively amplified.
TABLE 3 primers for amplifying target sequences with mutation sites
All primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Example 2 detection of samples Using the liquid chip for detecting CHEK1 Gene mutations described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH5.0) formulation (250 ml):
2 XTM hybridization buffer
| Reagent | Origin of origin | Final concentration | The dosage of each 250ml |
| 1MTris-HCl，pH8. | Sigma T3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
After filtration, the mixture was stored at 4 ℃.
The ExoSAP-IT kit was purchased from U.S. USB.
Biotin-labeled dCTP was purchased from Shanghai Biotechnology engineering services, Inc.
Firstly, DNA extraction of a sample:
the DNA to be detected is obtained by referring to the related method of DNA extraction in molecular cloning.
Second, PCR amplification of the sample to be tested
Three pairs of primers are designed, 3 amplification products respectively containing four common genotypes of the CHEK1 gene, namely C163T, C98A, G106A and A159G are amplified by multiplex PCR in one step, the sizes of the products are 337bp, 248bp and 361bp respectively, and the primer sequences (SEQ ID NO.25-30) are shown in the table 3.
Firstly, preparing a multiplex PCR primer working solution: respectively taking 100ul of primer stock solution of SEQ ID NO.25-30 into a 1.5ml microcentrifuge tube, and uniformly mixing to obtain the multiplex PCR primer working solution. The multiplex PCR reaction system is as follows:
the PCR amplification procedure was: 3min at 95 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃; storing at 4 deg.C for use.
Thirdly, enzyme digestion treatment of PCR product
1. Taking 7.5ul of the product after PCR reaction, adding 1ul of 10 XSAP buffer solution, 1ul of SAP enzyme and 0.5ul of Exo-I enzyme;
incubate at 2.37 ℃ for 15min, incubate at 80 ℃ for 15min, inactivate excess enzyme. The product after enzyme digestion is directly used for the subsequent ASPE primer extension reaction.
Site-specific primer extension reaction (ASPE)
The primer extension reaction was performed using the ASPE primers (table 1) designed as described above, and biotin-labeled dCTP was incorporated during the reaction, thereby allowing the products after the reaction to carry a plurality of biotin labels.
Firstly, preparing mixed ASPE primer working solution: respectively taking 10ul of wild type and mutant ASPE primer stock solution corresponding to the gene to be detected, adding 10mmol/L Tris Buffer to supplement to 200ul, and uniformly mixing to obtain the ASPE mixed primer working solution. The system for the ASPE reaction is as follows:
the reaction procedure is as follows: 2min at 96 ℃; 30 cycles of 94 ℃ for 30s, 54 ℃ for 1min, 72 ℃ for 2 min; storing at 4 deg.C for use.
Fifthly, hybridization reaction
1. Based on the designed ASPE primers, 8 corresponding coated microspheres (as described in example 1) were selected per set, and the microspheres were selected
The concentration of the spheres is 2.5 multiplied by 105Per ml;
2. 1ul of microspheres with each number are respectively taken and put in a 1.5ml microcentrifuge tube;
3. centrifuging the microspheres at a speed of more than or equal to 10000g for 1-2 min;
4. discarding the supernatant, resuspending the microspheres in 100ul of 2 XTM hybridization buffer, and mixing by vortex;
5. 25ul of the microsphere suspension was placed in the corresponding well of a 96-well filter plate, and 25ul of ddH was added to the control well2O；
6. Taking 5-25ul ASPE reaction solution into corresponding holes, and using ddH2O is complemented to 50 ul;
7. wrapping a 96-well plate with tin foil paper to avoid light, and incubating and hybridizing at 95 ℃ for 60s and 37 ℃ for 15 min;
8. centrifuging the hybridized microspheres for 2-5min at a speed of more than or equal to 3000 g;
9. removing supernatant, and suspending the microspheres in 75ul of 1 XTM hybridization buffer;
10. centrifuging the microspheres at a speed of more than or equal to 3000g for 2-5 min;
11. resuspend the microspheres in 75ul of 1 XTM hybridization buffer, add 15ul of streptavidin-phycoerythrin (SA-PE) at 10 ug/ml;
incubate at 12.37 ℃ for 15min and detect on Luminex instruments.
Sixthly, result detection and data analysis
And detecting the product after reaction by a Luminex series analytical instrument. The results of the measurements are shown in tables 4, 5 and 6.
The following requirements are placed on the fluorescence values (MFI) and data processing:
1. each locus needs to have at least one allele MFI greater than 300 and greater than 10 XPCR negative control MFI;
NET MFI ═ sample MFI-PCR negative control MFI (NET MFI less than 0 indicated as 0);
3. the mutation ratio is calculated according to the following formula from the data satisfying the above two conditions:
mutation ratio (mutant NET MFI + wild-type NET MFI)
4. A threshold (cut-off value) is empirically determined for the mutation ratio at each detection site to classify wild-type homozygotes, heterozygotes, and mutant homozygotes.
The method is used for synchronously detecting 4 mutation sites of the CHEK1 gene in 20 samples, and experimental data meet the requirements, so that the mutation ratio of the CHEK1 gene can be calculated. The threshold value (cut-off value) is set as follows: the mutation ratio ranging from 0% to 20% is regarded as wild type homozygote; 30% -70% are considered heterozygotes; 80% -100% are considered variant homozygotes. The results of the sequencing method detection and the liquid phase chip are compared, and the coincidence rate of the detection results of the typing method provided by the invention is calculated. The coincidence rate of the CHEK1 genotype detection result and the sequencing result of 20 samples detected by the method reaches 100%. Therefore, the liquid phase chip for detecting the CHEK1 gene mutation can accurately detect the type of the CHEK1 gene mutation site, and the result is stable and reliable.
TABLE 4 one of the sample test results (MFI)
TABLE 5 sample CHEK1 Gene mutation ratio (%)
TABLE 6 results of the analysis of the CHEK1 Gene mutation types
| Sample number | Liquid phase chip detection result | Sequencing results |
| 1 | Wild type | Wild type |
| 2 | Wild type | Wild type |
| 3 | Wild type | Wild type |
| 4 | Wild type | Wild type |
| 5 | Wild type | Wild type |
| 6 | Wild type | Wild type |
| 7 | 163TT | 163TT |
| 8 | Wild type | Wild type |
| 9 | 106GA | 106GA |
| 10 | Wild type | Wild type |
| 11 | Wild type | Wild type |
| 12 | 98AA | 98AA |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | Wild type | Wild type |
| 16 | 159GG | 159GG |
| 17 | Wild type | Wild type |
| 18 | Wild type | Wild type |
| 19 | Wild type | Wild type |
| 20 | Wild type | Wild type |
EXAMPLE 3 detection of CHEK1 Gene mutation site by liquid phase chip of different ASPE primers
Design of liquid phase chip preparation (tag sequence and Anti-tag sequence selection)
Taking a liquid phase chip for detecting mutation of C163T, C98A, G106A and A159G sites of CHEK1 genes as an example, specific primer sequences at the 3 'end of an ASPE primer are designed aiming at wild types and mutant types of C163T, C98A, G106A and A159G respectively, tag sequences at the 5' end of the ASPE primer are selected from SEQ ID NO.1-SEQ ID NO.8, and correspondingly, anti-tag sequences coated on microspheres and complementarily paired with the corresponding tag sequences are selected from SEQ ID NO.17-SEQ ID NO. 24. The specific design is shown in the following table (table 7). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 7 design of liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 21-40 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 8 sample test results and Gene mutation analysis
TABLE 9 sample test results and Gene mutation analysis
TABLE 10 sample test results and Gene mutation analysis
TABLE 11 sample test results and Gene mutation analysis
It can be seen from the above examples that, for liquid phase chips with different mutation sites, different tag sequences are applied to the ASPE primers, and the results are still stable and reliable, and specific data are omitted. When the tag sequence in the embodiment 1 is selected as the ASPE primer to match with the specific primer sequence, the effect is better (the signal to noise ratio is better), and the test group 1, the test group 5, the test group 8 and the test group 11 are referred to in the embodiment. Other different tag sequences are matched with the specific primer sequences, the results are the same as those of the example 2 and the example, and specific data are omitted.
Example 4 selection of primer sequences specific for the detection of a CHEK1 Gene mutation
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking a liquid phase chip for detecting mutation sites of CHEK1 genes C163T and G106A as an example, taking a forward or reverse complementary sequence of a target sequence where the mutation sites are located as a template, specific primer sequences at the 3' end of ASPE primers are designed aiming at wild types and mutant types of C163T and G106A respectively, and the specific primer sequences comprise a preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment 1 of the invention, as shown in Table 12. Wherein,the internal base is a mutation site.
TABLE 12 specific primer sequences
Taking the mutation site detection liquid phase chip of CHEK1 gene C163T and G106A as an example, different specific primer sequences are selected for C163T and G106A, and the tag sequence at the 5' end of the ASPE primer is fixed as the best effect sequence in example 1, and the ant-tag sequence corresponding to the best effect sequence is selected, and the specific design is shown in the following table (Table 13). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 13 design of liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 41-60 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 14 sample test results and Gene mutation analysis
TABLE 15 sample test results and Gene mutation analysis
As can be seen from this example, when the specific primer sequence in example 1 is selected as the ASPE primer to match with the tag sequence, the effect is better (the signal to noise ratio is better), see test group 13 and test group 16 in this example. Other different specific primer sequences derived from the forward or reverse complementary sequence of the target detection site are matched with the tag sequence, which is the same as the results of the embodiment 2 and the embodiment, i.e., the specific primer sequences described in the embodiments 1 and 2 are still better matched with different tag sequences, and specific data are omitted.
Other specific primer sequences aiming at different mutation sites are matched with tag sequences, and the results are the same as those of the embodiment 2 and the embodiment, namely, the specific primer selected in the embodiment 1 has better signal to noise ratio, better detection effect and specific data are omitted.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
- A liquid phase chip for detecting the mutation of CHEK1 gene is composed of a chip body with multiple liquid phases(A) Wild-type and mutant ASPE primer pairs designed for different mutation sites of the CHEK1 gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: at least one set selected from the group consisting of SEQ ID NO.9 and SEQ ID NO.10 for the C163T site, and SEQ ID NO.11 and SEQ ID NO.12 for the C98A site, SEQ ID NO.13 and SEQ ID NO.14 for the G106A site, and SEQ ID NO.15 and SEQ ID NO.16 for the A159G site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 8;(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;(C) a primer for amplifying a target sequence to be detected, which has a corresponding mutation site;the amplification primers are as follows: SEQ ID NO.25 and SEQ ID NO.26 for the C163T site, and at least one selected from the group consisting of SEQ ID NO.27 and SEQ ID NO.28 for the C98A site, and SEQ ID NO.29 and SEQ ID NO.30 for the G106A site, A159G site.
- 2. The liquid phase chip for detecting CHEK1 gene mutation of claim 1, wherein the ASPE primers are: at least one set selected from the group consisting of a sequence consisting of SEQ ID NO.1 and SEQ ID NO.9 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.10 for the C163T site, and a sequence consisting of SEQ ID NO.3 and SEQ ID NO.11 and a sequence consisting of SEQ ID NO.4 and SEQ ID NO.12 for the C98A site, a sequence consisting of SEQ ID NO.5 and SEQ ID NO.13 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.14 for the G106A site, and a sequence consisting of SEQ ID NO.7 and SEQ ID NO.15 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.16 for the A159G site.
- 3. The liquid phase chip for detecting a mutation in the CHEK1 gene of claim 1, wherein the chip comprises a chip body,(A) the ASPE primers are: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.9 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.10 for the C163T site, a sequence consisting of SEQ ID NO.3 and SEQ ID NO.11 and a sequence consisting of SEQ ID NO.4 and SEQ ID NO.12 for the C98A site, a sequence consisting of SEQ ID NO.5 and SEQ ID NO.13 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.14 for the G106A site, and a sequence consisting of SEQ ID NO.7 and SEQ ID NO.15 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.16 for the A159G site;(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;(C) the amplification primers are: SEQ ID NO.25 and SEQ ID NO.26 for the C163T site, SEQ ID NO.27 and SEQ ID NO.28 for the C98A site, and SEQ ID NO.29 and SEQ ID NO.30 for the G106A site, A159G site.
- 4. The liquid phase chip for detecting CHEK1 gene mutation according to any one of claims 1-3, wherein the spacer arm has 5-10T.
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| WO2002059354A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
| WO2005047533A1 (en) * | 2003-11-17 | 2005-05-26 | Tm Bioscience Corporation | Method of detecting mutations associated with thrombosis |
| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
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| WO2002059354A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
| WO2005047533A1 (en) * | 2003-11-17 | 2005-05-26 | Tm Bioscience Corporation | Method of detecting mutations associated with thrombosis |
| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
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| Comprehensive analysis of DNA repair gene polymorphisms and survival in patients with early stage non-small-cell lung cancer;Kim Min et al.;《Cancer Science》;20101130;第101卷(第11期);摘要、表1 * |
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